Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes.

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that key cytoskeletal proteins in DBD kidneys were proteolytically cleaved, generating peptide fragments, predominantly in grafts with suboptimal posttransplant function. Interestingly, α-actinin-4 and talin-1 proteolytic fragments were detected in brain death but not in circulatory death or living donor kidneys with similar donor characteristics. As talin-1 is a specific proteolytic target of calpain-1, we investigated a potential trigger of calpain activation and talin-1 degradation using human ex vivo precision-cut kidney slices and in vitro podocytes. Notably, we showed that activation of calpain-1 by transforming growth factor-ß generated proteolytic fragments of talin-1 that matched the degradation fragments detected in DBD preimplantation kidneys, also causing dysregulation of the actin cytoskeleton in human podocytes; events that were reversed by calpain-1 inhibition. Our data provide initial evidence that brain death donor kidneys are more susceptible to cytoskeletal protein degradation. Correlation to posttransplant outcomes may be established by future studies.

Donor age significantly influences the Raman spectroscopic biomolecular fingerprint of human pancreatic extracellular matrix proteins following collagenase-based digestion.

Despite the enormous advances in the field of clinical pancreatic islet transplantation over the past two decades, the human islet isolation procedure remains suboptimal. Islets are extracted (isolated) from the exocrine tissue of donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current isolation enzyme blends are ineffective at digesting pancreases from younger donors with low body mass indexes (BMI). However, age-related differences in pancreatic matrix digestion have not been studied in detail at the molecular level. To address this, we investigated ECM digestion in purified ECM proteins and in pancreatic tissue sections from younger (≤30 years; n = 5) and older (>55 years; n = 5) BMI matched donors, using Raman microspectroscopy (RMS). The Raman spectral profiles for purified collagens I, IV, VI and laminins were significantly altered following controlled enzyme treatment. Pancreatic cryosections were treated with Serva collagenase, NP, or the two enzymes combined, at clinically relevant concentrations. RMS demonstrated that the ECM at the islet-exocrine interface was differentially digested with respect to donor age. The action of collagenase was affected to a greater extent than NP. RMS is a powerful, marker-independent technology for characterising the human pancreatic ECM and demonstrating differences between donor types. Ongoing detailed studies using RMS will assist the development of donor-specific enzyme blends, increasing the overall success of human islet isolation and benefiting many people with type 1 diabetes worldwide. STATEMENT OF SIGNIFICANCE: Pancreatic islet transplantation is a minimally invasive treatment, which can reverse Type 1 Diabetes Mellitus (T1DM) in selected patients. Islets of Langerhans are extracted (isolated) from the exocrine tissue of human donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current enzymes are ineffective at digesting pancreases from younger donors. Using Raman microspectroscopy we demonstrate that donor age affects the enzymatic digestion of the pancreatic ECM at the molecular level. Collagenase activity is affected to a greater extent than NP. These findings will assist the development of donor-specific enzymes, thereby increasing the overall success of islet isolation and benefiting many people with T1DM worldwide.

Development of a Simple In Vitro Assay to Assess Digestion of the Extracellular Matrix of the Human Pancreas by Collagenase Enzyme Blends.

Despite huge advances in the field of islet transplantation over the last two decades, current islet isolation methods remain suboptimal, with transplantable yields obtained in less than half of all pancreases processed worldwide. Successful islet isolation is dependent on the ability of collagenase-based enzyme blends to digest extracellular matrix components at the islet-exocrine interface. The limited availability of donor pancreases hinders the use of full-scale islet isolations to characterize pancreas digestion by different enzyme components or blends, or allow the influence of inter-pancreatic variability between donors to be explored. We have developed a method that allows multiple enzyme components to be tested on any one pancreas. Biopsies of 0.5 cm3 were taken from seven standard (age ≥45) and eight young (age ≤35) pancreases. Serial cryosections were treated with Serva collagenase, neutral protease (NP), or the two enzymes together at clinically relevant concentrations. Following digestion, insulin and either collagen IV or laminin-α5 were detected by immunofluorescent labeling. Protein loss at the islet-exocrine interface was semi-quantified morphometrically, with reference to a control section. Differential digestion of the two proteins based on the enzyme components used was seen, with protein digestion significantly influenced by donor age. Treatment with collagenase and NP alone was significantly more effective at digesting collagen IV in the standard donor group, as was the NP mediated digestion of laminin-α5. Collagenase alone was not capable of significantly digesting laminin-α5 in either donor group. Combining the two enzymes ameliorated the age-related differences in the digestion of both proteins. No significant differences in protein loss were detected by the method when analyzed by two independent operators, demonstrating the reproducibility of the assay. The development of this simple yet reproducible assay has implications for both enzyme batch testing and identifying inter-donor digestion variability, while utilizing small amounts of both enzyme and human tissue.

Microplastics in the sediments of a UK urban lake.

While studies on microplastics in the marine environment show their wide-distribution, persistence and contamination of biota, the freshwater environment remains comparatively neglected. Where studies on freshwaters have been undertaken these have been on riverine systems or very large lakes. We present data on the distribution of microplastic particles in the sediments of Edgbaston Pool, a shallow eutrophic lake in central Birmingham, UK. These data provide, to our knowledge, the first assessment of microplastic concentrations in the sediments of either a small or an urban lake and the first for any lake in the UK. Maximum concentrations reached 25-30 particles per 100 g dried sediment (equivalent to low hundreds kg-1) and hence are comparable with reported river sediment studies. Fibres and films were the most common types of microplastic observed. Spatial distributions appear to be due to similar factors to other lake studies (i.e. location of inflow; prevailing wind directions; propensity for biofouling; distribution of macroplastic debris) and add to the growing burden of evidence for microplastic ubiquity in all environments.

Hematological and serum biochemical reference values and cohort analysis in the Gilbert's potoroo (Potorous gilbertii).

Hematology and serum biochemistry blood values are tabulated for Australia's most critically endangered mammal, the Gilbert's potoroo (Potorous gilbertii). Significant differences were found between origin (captive or wild individuals) and age (subadult or adult) of animals. Sex, and presence or absence of Treponema infection, had minimal significance on blood values. Typical cell morphology is discussed, and hemoparasite examination identified Theileria spp. and Breinlia spp. Eighty samples were collected from a population of only 35 individuals, reflective of a population census rather than of a study reliant on statistical extrapolation. These reference ranges and findings will assist in the ongoing health management of this critically endangered species. hematology, biochemistry, marsupial, Gilbert's potoroo, Potorous gilbertii.

An outbreak of mycobacteriosis in Gouldian finches caused by Mycobacterium peregrinum.

An outbreak of mycobacteriosis was detected in an aviary containing Gouldian finches (Erythrura gouldiae) and golden shouldered parrots (Psephotus chrysopterygius). Affected birds developed granulomatous lesions, usually of the liver and intestine. Mycobacterium peregrinum, a species of the Mycobacterium fortuitum group, was cultured on pooled samples of intestinal tract from 31 euthanized finches. These rapid-growing mycobacteria are saprophytic organisms that are generally not associated with clinical disease in immunocomponenet hosts. This is the first report of mycobacteriosis in finches implicating M peregrinum as a causative agent.

A splendid tree frog with edema syndrome and intestinal adenocarcinoma.

A splendid tree frog (Pelodryas splendida) presented with subcutaneous edema extending along its dorsum from head to vent, which resolved with improvement of ambient temperature and humidity conditions in its enclosure. Four months later, this same frog presented in a moribund state with intracoelomic fluid accumulation. An intestinal adenocarcinoma, a rarely reported neoplasm in amphibians, was diagnosed post mortem. Neoplasia is one of a number of causes of edema syndrome, which is a nonspecific response to disease and debility in anurans.